C. trachomatis is the most common sexually transmitted bacterial pathogen in the US. Chlamydia infections appear to have adverse effects on women's health including complications of pregnancy, pelvic inflammatory disease, infertility, and cervical infections. The objectives of this project are to define the epidemiology, risk factors, transmission kinetics and pathogenesis of Chlamydia trachomatis infections in different population settings and different disease states using new molecular amplification assays. To address this objective, we have continued screening using non-invasive samples with molecular amplification assays for C. trachomatis and documented high rates of infection in sexually active adolescents in diverse cultural settings in six countries. Prevalence rates of chlamydia ranged from 3.9% in rural villages in Zimbabwe and Uganda, 11.1% in St. Petersburg, Russia, 5.5% in Lima, Peru, 8.8% in Fuzhou, China, and 1.2% in Chennai, India. STD clinics in Baltimore routinely run 20% for men, 11.4% for women and as high as 14% for high school students. We have determined that in high school students 25% of infected students get re-infected within one year. The documented high rates of chlamydia in these countries raises serious concerns about the resurgence of STDs that may reflect a rise in high-risk behavior which may subsequently lead to further HIV transmission. In a study of adolescent males in the US, we determined the cost effectiveness of chlamydia screening on admission to detention centers. Chlamydia prevalence in the population was 4.8% and the average number of female sexual partners per infected male was 1.6. We determined that universal screening by nucleic acid amplified test was the most cost-effective strategy preventing 37 more cases of pelvic inflammatory disease and three more cases of epididymitis than selective screening. With the implementation of universal screening we have performed a new study comparing self-administered vaginal swabs, cervical swabs, and first-catch urines for the diagnosis of chlamydial and gonorrhea infections in women from STD clinics in Baltimore. Specimens from 321 women were compared. Results showed that a self-administered vaginal swab detected the most infections; 10.8% prevalence compared to cervical swabs (10.6%) or urine (10.2%) indicating that self-administered vaginal swabs were equivalent or even better than cervical or urine samples for the detection of chlamydia. For gonorrhea, the vaginal swab performed better that cervical or urine samples; vaginal swab 5.0% prevalence, cervical 3.8%, and urine 4.3% prevalence. This study and others we have previously reported demonstrate that vaginal swabs are appropriate specimens for diagnosing chlamydial genital tract infection by amplified diagnostic assays. Non-invasive screening for chlamydia can be applied in different clinical and non-clinical sites thereby increasing the size of the population to be screened. We have additionally developed research realtime PCR assays for other STDs, such as Trichomonas vaginalis, Mycoplasma genitalium, and the LGV serovars of Chlamydia trachomatis. We have screened for C. trachomatis infections in a surgical/antibiotic treatment study of trachoma in Ethiopia and Tanzania by performing PCR studies as outcome measures of these interventions. In support of world-wide clinical trials for safety and efficacy of vaginal microbicides we have recently tested their potential inhibitory effect in urine samples on the ability of molecular amplification assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae. Some older molecular assays fail to detect these organisms when they are in urine with the microbicides, but for the majority, most amplified assays are not affected by the presence of microbicides in urine samples.